Envision labelled polymer hrp anti rabbit

PTEN expression was assessed by immunohistochemistry (IHC) on freshly cut 4μm tissue sections of FFPE specimens that were deparaffinized, followed by heat-mediated antigen retrieval using 0.1M Tris-HCL (pH 9.0) buffer. To decrease nonspecific binding, sections were first treated with 3% hydrogen peroxide solution. Tissues were then incubated for 1 hour at room temperature with rabbit polyclonal PTEN (D4.3) antibody (Cell Signaling, Beverly, MA) at a 1:100 dilution in SignalStain antibody diluents (Cell Signaling), followed by incubation with Envision Labelled Polymer-HRP Anti-Rabbit (Dako, Carpenteria, CA), DAB+ solution (Dako), and DAB Sparkle Enhancer (Biocare, Concord, CA) for detection. Immunohistochemical results were evaluated independently by two pathologists (A.C and S.H.) who were blinded to the results of genetic analysis and patient outcome. As a specimen’s internal control, stromal cells were assessed for positive staining; samples devoid of staining in the internal controls were considered uninterpretable. Each section was scored for percent positivity (0–100%) and level of intensity (0–3). The H-score of each sample was calculated by multiplying the percentage and intensity scores.